Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa.

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.

Synthesis, SAR and in vivo activity of novel thienopyridine sulfonamide pyrrolidinones as factor Xa inhibitors.

Thienopyridine sulfonamide pyrrolidinones were found to be potent and selective inhibitors of the coagulation cascade enzyme factor Xa. SAR studies led to several compounds that were selected for further in vivo investigation. These novel aryl binding pocket moieties represent a structural modification to a series of fXa inhibitors. Several compounds proved to be efficacious i.v. antithrombotic agents.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Cardiovascular and metabolic effects of adenosine A1-receptor agonists in streptozotocin-treated rats.

Because one manifestation of diabetes is an enhancement of the lipolytic process, we evaluated the antilipolytic effects of adenosine A1 agonists in vitro and in vivo in streptozotocin (STZ)-treated diabetic rats. In vitro, we examined the responses to norepinephrine (NE) and adenosine deaminase (ADA), as well as several adenosine A1 agonists, in isolated adipocytes from normal and diabetic rats. Both NE and ADA caused dose-dependent stimulation of lipolysis, elevating glycerol release twofold to threefold over baseline. The sensitivity to both NE and ADA was significantly enhanced in adipocytes from STZ-treated as compared with normal rats. N-5'-ethyl-N(6)(cyclopentyl) adenosine-5'-uronamide (RG14202) was by far the most potent A agonist in inhibiting NE-stimulated lipolysis [50% effective concentration (EC50): 0.014 +/- 0.0008 nM), approximately 1 and 2 log units more potent than N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyl-2'-O-methyladenosine (SDZ WAG 994), respectively. In vivo we established a model for evaluating the therapeutic utility of adenosine A1 agonists, emphasizing duration of action. In STZ rats instrumented with telemetry transmitters, the metabolic effects of CPA, RG14202, and SDZ WAG 994 were assessed 6 h after oral administration. Under those conditions, RG14202 and SDZ WAG 994, but not CPA, significantly reduced triglycerides (TRIs) and TRI/free fatty acids (FFAs), respectively. However, all three A1 agonists dose-dependently reduced mean arterial pressure (MAP) and heart rate (HR) concurrently. Thus adenosine A1 agonists can inhibit lipolysis in vitro and in vivo; however, oral administration produces long-lasting beneficial metabolic effects only at doses that also produce a significant bradycardia.

Cardiovascular and antilipolytic effects of the adenosine agonist GR79236.

Adenosine is known to produce cardiovascular effects such as bradycardia and hypotension via activation of myocardial (A1) and vascular (A2) receptors and antilipolytic effects through activation of adipocyte (A1) receptors. We established the cardiovascular and antilipolytic profile of the adenosine A1 agonist GR79236 (N6-[(1S,trans)-2-hydroxycyclopentyl]-adenosine) and compared it with CPA (N6-cyclopentyl-adenosine). GR79236 was approximately 3-fold less potent than CPA in inhibiting in vitro lipolysis. In conscious rats, both agents were shown to have antilipolytic and glucose-lowering properties. In rats instrumented with telemetry transmitters, orally administered CPA was one log unit more potent than GR79236 as a hypotensive and bradycardiac agent. In summary, GR79236 is an A1-selective adenosine agonist which reduces heart rate and mean arterial pressure and produces decreased plasma lipids and glucose levels.

In vitro and in vivo characterization of an A1-selective adenosine agonist, RG14202.

In this report, we demonstrate that the adenosine agonist N-5'-ethyl-N6-(cyclopentyl) adenosine-5'-uronamide (RG14202) is a vasorelaxant in porcine coronary arterial rings (EC50 = 0.37 +/- 0.054 microM; n = 19). This vasorelaxation (VR) occurs despite RG14202 being 275-fold selective for the rat brain A1 receptor. VR in response to RG14202 was attenuated markedly by the nonselective adenosine antagonist CGS15943, whereas 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 antagonist, had only a small inhibitory effect. In contrast, the potassium channel blocker glybenclamide attenuated RG14202-induced VR markedly (85-fold), indicating that modulation of potassium channels is likely involved. In carotid arterial rings, RG14202 was approximately 5 times less potent than in the coronary artery, suggesting that this compound may be more selective for the coronary vasculature. In anesthetized rats, i.v. administration of RG14202 caused a significant decrease in mean arterial pressure only at the highest dose (3 micrograms/kg). In comparison, heart rate was decreased dose-dependently with maximal changes at 3 micrograms/kg. Both the depressor and bradycardic responses could be antagonized with CGS15943. RG14202 increased renal, but had no effect on mesenteric or hindquarter vascular resistance. Glybenclamide pretreatment (20 mg/kg) did not significantly alter the effects of RG14202 on heart rate or regional vascular resistances; however, the depressor response to RG14202 was attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of EGF-induced vasoconstriction in isolated rabbit aortic rings with the tyrosine kinase inhibitor RG50864.

The tyrosine kinase inhibitor RG50864 attenuated EGF-induced tension development dose-dependently. Similarly, contractions to a phorbolester were affected by 10 microM RG50864. In comparison, vasoconstrictor activities of norepinephrine, endothelin and Bay K 8644 remained unaltered. Western blots using antiphosphotyrosine antibodies, revealed a time-dependent increase in EGF-induced EGF-receptor autophosphorylation as well as tyrosine phosphorylation of a 55 kDalton cytosolic protein. While the extent of EGF-receptor autophosphorylation remained unaltered in the presence of 10 microM RG50864, phosphorylation of the 55 kD band was decreased two-fold. In summary, in rabbit aorta RG50864 is an inhibitor of EGF-induced vasoconstriction; this inhibitory effect does not appear to be mediated through inhibition of EGF-receptor autophosphorylation but may involve a 55 kD cytosolic protein substrate.

Protein kinase C and vascular smooth muscle contractility: effects of inhibitors and down-regulation.

We have compared two different methods of attenuating protein kinase C (PKC) activity in vascular smooth muscle. First, the effects of two purported PKC inhibitors, staurosporine (stauro) and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], were examined on contractility of isolated, intact canine femoral artery. In arterial rings stauro was equipotent in relaxing contractions induced by phenylephrine (PE), phorbol-12,13-dibutyrate (PDBu) and KCl (IC50, 0.31 +/- 0.19; 0.35 +/- 0.2; and 0.34 +/- 0.16 microM). H-7, in comparison, was markedly less potent than stauro (IC50, 0.67 +/- 0.2, 2.33 +/- 0.24; and 6.5 +/- 5.5 microM for PE, PDBu and KCl, respectively). Pretreatment of tissues with 1 microM stauro suppressed tension development almost completely when PE and PDBu were the contractile agonists, and partially in K(+)-depolarized rings. H-7, in contrast, had no inhibitory effect on agonist-induced contraction. Neither basal nor K(+)-stimulated calcium influx was affected by 10 microM stauro. Second, prolonged exposure of canine carotid arterial rings to PDBu (1-100 nM for 24 hr), a means of depleting PKC from the tissue, caused dose-dependent attenuation of agonist-induced contractions. Preincubation with 100 nM PDBu caused complete inhibition of tension induced by norepinephrine (NE) and serotonin and partial inhibition of PDBu- and KCl-induced contractions. Lowering the concentration of PDBu during preincubation to 30, 10 or 1 nM reduced markedly the inhibitory effects. The inactive phorbolester 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD) had no effect on agonist-induced contractions. PKC activity was determined in rings contracted isometrically with PDBu or NE after prolonged exposure to vehicle, 4 alpha-PDD or PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)